In recent years, the number of obese subjects is going on increasing throughout the world. It is no exaggeration to say that “obesity” is a root cause of all lifestyle-related diseases. Various obesity-related diseases (e.g., diabetes mellitus, arteriosclerosis caused by hyperlipidemia or hypertension, and ischemic heart disease or cerebrovascular disorder caused thereby) are the major causes of death in developed countries including Japan. In particular, Japanese people are apt to develop diabetes mellitus even if the degree of obesity is low, compared to Western people, and therefore need a countermeasure against obesity with higher urgency. In emerging countries such as China, Brazil, India, and Russia, the rate of increase in obese subjects is further significant and has become a large social issue.
The basic treatment for obesity is an improvement of lifestyle based on diet therapy and exercise therapy, but it is not easy for obese subjects to reduce weight. The number of obesity patients who cannot obey diet is not small. In addition, it is difficult to perform exercise therapy in many cases due to various diseases associated with obesity (e.g., osteoarthritis, diabetic gangrene, and heart failure).
At the same time, as described by the words “big eater who stays thin”, it has been indicated from the past that there is an “important factor” other than excessive eating and lack of exercise as a cause of obesity.
In conventional research on obesity, white adipose tissue (WAT) has been mainly studied.
Recently, it was accidentally revealed in data analysis of nuclear medicine examination (PET/CT) that human also has brown adipose tissue (BAT), which was believed to be present in rodents only (Non-Patent Literature 1). Similar reports have been done successively, and an inverse correlation between the amount of BAT and obesity/onset of metabolic syndrome has been also reported. Today, BAT is recognized as significantly important tissue for assessing the pathological conditions of obesity.
BAT and WAT are also developmentally different from each other. BAT is already formed in the fatal stage, whereas WAT is mainly developed after birth. It is also known that in large mammals including human, the majority of BAT disappears (physiological disappearance) within two days after birth. The mechanism of this is not known at all. The remaining BAT also gradually decreases with aging, of which mechanism is also unknown.
Accordingly, in order to correctly comprehend BAT, it is also important to elucidate the mechanism of the physiological disappearance observed after birth and the disappearance associated with aging. In particular, it should be noticed that small mammals such as mice are not useful for elucidating the mechanism of physiological disappearance.
WAT is an energy storage tissue for storing fat, whereas BAT is an energy production tissue for actively burning fat. The both have absolutely different characteristics in cellular morphology and gene expression.
Morphologically, white adipocytes contain large unilocular lipid droplets and are poor in mitochondria (a small number of mitochondria are present only at the periphery of the nucleus), of which morphology shows segmentation as reflection of a low oxidative phosphorylation activity. On the other hand, brown adipocytes contain small multilocular lipid droplets and are abundant in mitochondria (localizing at the peripheries of the lipid droplets), of which the morphology shows string-like fusion being long lengthwise as reflection of a high oxidative phosphorylation activity and a large number of ladder-type cristae developed intracellularly.
Regarding the gene expression, for example, WAT is characterized by the expressions of resistin and phosphoserine aminotransferase 1 (PSAT1), whereas BAT is characterized by the expressions of elongation of very long chain fatty acids-like 3 (ELOVL3), cell death-inducing DFFA-like effector A (CIDE-A), peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferative activated receptor gamma coactivator 1α (PGC1α), cytochrome C (Cyt-c), epithelial V-like antigen (EVA1), and neurotrophic tyrosine kinase receptor type 3 (NTRK3), in addition to uncoupling protein 1 (UCP1) and PR domain containing 16 (PRDM16), etc.
Among them, UCP1 has an activity of uncoupling the oxidative phosphorylation and ATP production in mitochondria and thereby has an effect of blocking the active oxygen production inevitably associated with oxidative phosphorylation. That is, BAT shows a noteworthy effect of removing oxidative stress concomitant with biological activity through expressing UCP1.
It is also known that WAT and BAT show conflicting physiological effects in vivo. WAT induces oxidative stress by the hypertrophy of cells due to excessive accumulation of fat. As a result, inflammation of adipose tissue is caused to induce insulin resistance at an individual level due to influence of, for example, inflammatory cytokines. However, in BAT, since UCP1 is highly expressed, oxidative stress is not induced, and the insulin sensitivity at an individual level is enhanced.
Thus, BAT has not only an anti-obesity activity but also an activity of improving insulin resistance and is therefore expected to have preventive and therapeutic effects on type 2 diabetes mellitus.
In addition, it has been recently reported that BAT actively uptakes lipids from blood and burns and actively consumes them to show a therapeutic effect on hyperlipidemia (Non-Patent Literature 2).
In a coronary artery bypass surgery, transplantation of WAT of a patient into the surgery site before the surgery improves the results at least in the short term. However, many patients who need coronary artery bypass surgeries have already developed metabolic syndrome. Consequently, vascular restenosis after surgery is concerned in the long term, due to initiation of inflammatory reaction and induction of oxidative stress in the transplanted adipocytes. It is also known that in coronary artery stenosis cases, a large amount of WAT is actually present around the coronary artery.
If BAT, which does not induce oxidative stress, can be transplanted into the site of coronary artery bypass surgery, an improvement in the long-term results can be expected.
Thus, BAT, which is expected to have therapeutic effects on obesity, insulin resistance, type 2 diabetes mellitus, and hyperlipidemia and an effect of improving the result of coronary artery bypass surgery, is significantly important and valuable tissue for complete cure of various diseases associated with metabolic syndrome.
Unfortunately, the occurrence, growth mechanism, functional regulation, and other factors of BAT in human are still unclear in many points. Furthermore, all of adipokines that are known to be involved in metabolic regulation as adipose tissue-derived hormones were identified from WAT, and no “BAT-specific adipokine” that can be expected to show anti-obesity and metabolism-improving activities superior to those of existing adipokines has been identified.
Thus, important findings relating to BAT have not been obtained yet. This is caused by that BAT specimens from normal volunteers are hardly obtained by the following four reasons: 1) in order to identify the positions of BAT, PET/CT inspection, which causes a large quantity of radiation exposure, is necessary; 2) BAT in not all subjects can be visualized by the PET/CT inspection; 3) BAT is scattered in multiple sites (e.g., posterior cervical region and the side of each thoracic vertebra) in the human adult body, and the quantity of BAT is not high (not higher than 300 g in total); and 4) there are no sufficient data for evaluating demerits (e.g., an increase in risk of onset of metabolic syndrome) caused by removal of BAT being such minute tissue.
In order to overcome these problems and supply a sufficient amount of brown adipocytes for the use thereof for research purposes and clinical application (such as cell therapy), it is significantly useful to produce brown adipocytes from pluripotent stem cells having both self-replication ability and pluripotent differentiation ability.
Examples of most generally useful human pluripotent stem cells include human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells. However, production of brown adipocytes using these human pluripotent stem cells has not been achieved successfully yet.
There are reports on the production of brown adipocytes from somatic stem cells present in mouse bone marrow, skin, and adipose tissue and the production of brown adipocytes from mouse ES cells (Patent Literature 1). However, the in vitro test for confirming the production of brown adipocytes is performed by detecting the expression of message of a UCP1 gene only, and the expressions of other members of the gene cluster (e.g., PRDM16, ELOVL3, CIDE-A, and PPARα) important for expressing the brown adipocyte functions are not investigated. In addition, evaluation from the cellular morphological viewpoints is not performed at all. Accordingly, the quality of the produced brown adipocyte is questionable.
In addition, it is absolutely unclear whether or not this method is applicable to human pluripotent stem cell. Considering that mice have a large amount of BAT and that conversion of WAT into BAT by chronic cold stimulation is frequently observed in mice, it is significantly difficult to believe that the results in mice are directly applicable to human.
Furthermore, as described above, the mechanism of physiological disappearance observed in large mammals including human cannot be elucidated using the mouse stem cell-derived brown adipocytes produced by the above-mentioned method.
There is also a report on that brown adipocytes are produced by culturing preadipocytes collected from mouse BAT or 10T1/2 cell line derived from mouse fetus in a medium containing BMP7 (Non-Patent Literature 3). However, it is absolutely unclear whether or not this method is applicable not only to mouse pluripotent stem cells but also to human pluripotent stem cells.
There is a report on that brown adipocytes were produced by introducing two genes (CCAAT/enhancer binding protein β (C/EBPβ) and PRDM16) into human neonatal fibroblasts (Non-Patent Literature 4). However, considering the lifetime of human fibroblasts, it is difficult to prepare a large amount of brown adipocytes for the purpose of providing a research material or a cell therapy tool. Since these brown adipocytes are forcedly produced by introducing genes, these brown adipocytes are not suitable for use in elucidation of the mechanism of the above-described physiological disappearance.
Accordingly, development of a technology that is applicable to human pluripotent stem cells including human ES cells and human iPS cells and can produce brown adipocytes from pluripotent stem cells without forced induction of differentiation by gene introduction is an urgent need for facilitating basic research on various diseases associated with metabolic syndrome, which is a large social issue in developed countries and emerging countries, and for developing preventive and therapeutic methods.